Morphology and Multigene Phylogeny Revealed Three New Species of Helminthosporium (Massarinaceae, Pleosporales) from China

Saprobic hyphomycetes are highly diverse on plant debris. Over the course of our mycological surveys in southern China, three new Helminthosporium species, H. guanshanense sp. nov., H. jiulianshanense sp. nov. and H. meilingense sp. nov., collected on dead branches of unidentified plants, were introduced by morphological and molecular phylogenetic analyses. Multi-loci (ITS, LSU, SSU, RPB2 and TEF1) phylogenetic analyses were performed using maximum-likelihood and Bayesian inference to infer their taxonomic positions within Massarinaceae. Both molecular analyses and morphological data supported H. guanshanense, H. jiulianshanense and H. meilingense as three independent taxa within Helminthosporium. A list of accepted Helminthosporium species with major morphological features, host information, locality and sequence data was provided. This work expands our understanding of the diversity of Helminthosporium-like taxa in Jiangxi Province, China.


Introduction
Hyphomycetes, a group of anamorphic Ascomycota, are highly diverse in aquatic and terrestrial habitats and distributed worldwide on many natural substrates such as plant tissues, wood and bark, dung, insects and other arthropods and other fungi including lichens [1]. More than 30,000 species of asexual fungi are recorded worldwide, with 2500 hyphomyceteous genera [2,3]. The most comprehensive occurrence of this group is in the northern temperate regions, with little recorded in tropical and subtropical areas [1,3]. China is considered an important reservoir of biodiversity by the Convention on Biological Diversity. However, more research on fungal diversity in China is needed.
Jiangxi, located in the southeast of China, is one of the most biodiverse provinces. Its preserved superior ecological environment, humid subtropical climate and abundant plant resources would suggest that the province also has great fungal diversity. During a survey of saprobic hyphomycetes from plant debris in this province, three interesting hyphomycetes belonging to the genus Helminthosporium (Massarinaceae, Pleosporales) were collected on dead branches. Based on the multi-locus phylogenetic analysis and morphological examination, they are introduced as new to science in the present study.

Sample Collection, Isolation and Morphology
Samples of dead branches were collected from humid environments and river banks in the subtropical forests of Jiangxi Province, China, and placed in Ziploc™ plastic bags. Samples were processed and examined following the methods described in Ma et al. [35]. Colonies on decaying wood surfaces were examined and visually observed with a stereomicroscope (Motic SMZ-168, Xiamen, China) from low (0.75 times) to high (5 times) magnification. Fresh colonies were picked with sterile needles at a stereomicroscope magnification of 5 times, placed on a slide with a drop of lactic acid-phenol solution (lactic acid, phenol, glycerin, sterile water; 1:1:2:1, respectively), then placed under an Olympus BX 53 light microscope fitted with an Olympus DP 27 digital camera (Olympus Optical Co., Tokyo, Japan) for microscopic morphological characterization. The tip of a sterile toothpick dipped in sterile water was used to capture the conidia of the target colony directly from the specimen; the conidia were then streaked on the surface of potato dextrose agar (PDA; 20% potato + 2% dextrose + 2% agar, w/v) and incubated in an incubator at 25 • C overnight. The single germinated conidia were transferred to fresh PDA plates following the method of Goh [36] and incubated in an incubator at 25 • C. Culture characteristics were examined and recorded after 3 days and later at regular intervals for 3 days. Colony colors were assessed according to the charts of Rayner [37]. All fungal strains were stored in 10% sterilized glycerin at 4 • C for further studies. The studied specimens and cultures were deposited in the Herbarium of Jiangxi Agricultural University, Plant Pathology, Nanchang, China (HJAUP). The names of the new taxa were registered in Index Fungorum [3].

DNA Extraction, PCR Amplification and Sequencing
Genomic DNA was extracted from fungal mycelia grown on PDA, using the Solarbio Fungi Genomic DNA Extraction Kit (Solarbio, Beijing, China) following the manufacturer's protocol. DNA amplification was performed by polymerase chain reaction (PCR) using the respective loci (ITS, LSU, SSU, TEF1 and RPB2). The following primer sets were used for these genes: ITS: ITS5/ITS4 [38]; LSU: 28S1-F/28S3-R [39]; SSU: 18S-F/18S-R [39]; TEF1: EF1-983F/EF1-2218R [28,40] and RPB2: RPB2-5F2 [41]/fRPB2-7cR [42]. The amplifications were performed in a 25 µL reaction volume containing 12.5 µL of 2 × Power Taq PCR MasterMix, 1 µL of each forward and reverse primer, 1 µL of DNA template and 9.5 µL of ddH 2 O. The PCR thermal cycle program for ITS, LSU, SSU and TEF1 amplification was as follows: 95 • C for 3 min, followed by 35 cycles of denaturation at 94 • C for 15 s, annealing at 55 • C for 15 s, elongation at 72 • C for 30 s and finally extended at 72 • C for 5 min. Regions of RPB2 were amplified with annealing at 59 • C for 15 s, elongation at 72 • C for 2 min and others consistent with the above procedure. The PCR products were checked on 1% agarose gel electrophoresis stained with ethidium bromide. Purification and sequencing of PCR products were carried out by Beijing Tsingke Biotechnology Co., Ltd. China. New sequences generated in this study were deposited in the NCBI GenBank (www.ncbi.nlm.nih.gov, accessed on 5 January 2023; Table 1).

Phylogenetic Analyses
The newly generated sequences together with other sequences obtained from GenBank (Table 1) were aligned using MAFFTv.7 [43] on the online server (http://maffTh.cbrc.jp/ alignment/server/, accessed on 5 January 2023), and optimized manually when needed. Phylogenetic analyses were conducted individually for each locus at first and then for a combined dataset of five gene loci (ITS, LSU, SSU, TEF1 and RPB2). The tandem sequences of ITS, SSU, LSU, TEF1 and RPB2 were obtained by Phylosuite software v1.2.1 [44] under "Concatenate Sequence", and absent sequence data in the alignments were treated with a question mark as missing data. The best-fitting nucleotide substitution models for each alignment dataset were selected using ModelFinder [45]. Maximum-likelihood (ML) and Bayesian inference (BI) were used to analyze the dataset after splicing. Maximum-likelihood phylogenies were inferred using IQ-TREE [46] under an Edge-linked partition model for 10,000 ultrafastbootstraps [47]. The optimal ML tree search was conducted with 1000 separate runs using the default algorithm of the program from a random starting tree for each run. The best-fit model was TIM3 + F + R3 for TEF1, TIM2e + I + G4 for ITS, TNe + R4 for LSU, TN + F + I + G4 for RPB2 and K2P + R2 for SSU. Bayesian inference phylogenies were inferred using MrBayes 3.2.6 [48] under a partition model (2 parallel runs, 2,000,000 generations), in which the initial 25% of sampled data were discarded as burn-in. The best-fit model was GTR + F + I + G4 for RPB2, ITS and LSU, GTR + F + G4 for TEF1 and HKY + F + G4 for SSU. ModelFinder [45] was used to select the best-fit partition model (Edge-linked) using BIC criterion. These trees were visualized using FigTree v. 1.4.4 (http://tree.bio.ed.ac.uk/software/figtree, accessed on 16 January 2023), with editing and typesetting using Adobe Illustrator CS v. 5.

Molecular Phylogeny
In this study, five gene regions of ITS, LSU, SSU, TEF1 and RPB2 were obtained successfully except for H. jiulianshanense, which lack SSU and TEF1 sequences. Phylogenetic relationships of three Helminthosporium species were assessed in the combined analysis  The ML and BI bootstrap support values above 75% and 0.90 are given above the nodes. The tree is rooted to Cyclothyriella rubronotata (TR) and C. rubronotata (TR9). Strains from the current study are in bold. Some branches were shortened according to the indicated multipliers.

Taxonomy
Helminthosporium guanshanense Y.F. Hu and Jian Ma, sp. nov., Figure 2. Indexfungorum number: IF900239. Etymology: The name refers to Guanshan Nature Reserve, the locality where the fungus was collected.
Holotype: HJAUP M1022. The ML and BI bootstrap support values above 75% and 0.90 are given above the nodes. The tree is rooted to Cyclothyriella rubronotata (TR) and C. rubronotata (TR9). Strains from the current study are in bold. Some branches were shortened according to the indicated multipliers.
Culture characteristics: Colony on PDA reaching 55-65 mm diam. after 4 weeks in an incubator under dark conditions at 25 °C, irregular circular, surface reddish dark brown with gray white in the center and gray mat of aerial hyphae at the margin; reverse rosybrown with black dots and pale brown periphery.
Culture characteristics: Colony on PDA reaching 70-78 mm diam. after 4 weeks in an incubator under dark conditions at 25 • C, irregular circular, surface velvety, with dense, dark brown mycelium plus white patches of aerial hyphae, and becoming sparser towards the edge; reverse gray with sparser black patches in the center.

Discussion
The establishment of Helminthosporium was based on morphological studies. More than 770 epithets for Helminthosporium have been listed in Index Fungorum [3]. Members in the genus mainly occur in the asexual morph, usually forming effuse, hairy colonies on decaying leaf or twig litter. The generic concept of Helminthosporium is based on the characteristics of asexual morph and is mainly characterized by distinct, determinate or percurrently extending conidiophores with a well-defined small pore at the apex and/or laterally beneath the septa and tretic, integrated, terminal or intercalary conidiogenous cells that produce solitary (rarely in short chains), clavate or obclavate, distoseptate conidia usually with a distinct dark brown to black scar at the base [1,4,7,11,24,50,51]. Voglmayr and Jaklitsch [24] transferred four Corynespora species to Helminthosporium based on molecular phylogenetic analyses, which led to the characters delineating the genus Helminthosporium also covering the criteria of Corynespora. The traditional distinction between monotretic vs. polytretic conidiogenous cells for separating Corynespora and Helminthosporium is shown to be insignificant in a phylogenetic context.
The taxonomic history of the genus Helminthosporium is complex. Many graminicolous taxa conventionally named as "Helminthosporium" species have been reclassified into the genera Bipolaris, Curvularia, Drechslera and Exserohilum [8], and several lignicolous species were recently transferred to Ellismarsporium, Mirohelminthosporium, Stanhughesiella, Varioseptispora and other genera due to their atypical features in Helminthosporium [11][12][13]. Konta et al. [34] listed 216 Helminthosporium species based on records from Species Fungorum, but many species are identified based only on morphological studies, and only 33 species have sequence data so far. Morphological comparison is important for fungal identification, but species identification only based on morphological studies is not comprehensive [31]. There is presently a strong tendency to evaluate previous described Helminthosporium species by molecular methods. Thus, resurrection of the genus Helminthosporium and studying their diversity and biology by a morpho-molecular approach are urgently necessary, which may be helpful to clarify the taxonomic status of many doubtful species and some important plant pathogens [23,28].
The genus Helminthosporium has a worldwide distribution with species recorded from a wide range of hosts [23,24,30,34,[51][52][53][54][55]. However, the number of Helminthosporium species is very confusing in the recent monograph [24,28,[56][57][58]. For example, Kirk et al. [56] recorded in the Dictionary of the Fungi that the genus comprises c. 35 species. Wijayawardene et al. [57,58] respectively estimated the genus including c. 40 and 416 species. Voglmayr and Jaklitsch [24] approximated the number of taxa accepted in Helminthosporium is about 46. In addition, Voglmayr and Jaklitsch [24] synonymised Exosporium with Helminthosporium and evaluated 17 Helminthosporium species by morphological and molecular systematic analysis. Konta et al. [34] listed 216 Helminthosporium species based on the records of Species Fungorum 2021. Considering that the number of Helminthosporium species does not match and many subsequent authors followed Siboe et al.'s [10] treatment, 63 species are currently accommodated in this genus. A checklist for these 63 Helminthosporium species, including major morphological features, host information, locality and sequence data, is provided in Table 2. Most of these are commonly collected from leaves and decaying wood in terrestrial habitats [22,24,30,34], and only two species, H. aquaticum Hong Y. Su, Z.L. Luo and K.D. Hyde and H. submersum Z.L. Luo, N. Zhao, K.D. Hyde and H.Y. Su, are recorded in freshwater habitats [23,28]. Thus, large-scale surveys of fungal resources in aquatic and terrestrial habitats with different geographic regions, ecological environment, vegetation type and climatic conditions will contribute to the knowledge of the fungal diversity and to a better understanding of the doubtful species, further clarifying their taxonomic status by phylogenetic analyses.